A Homozygous Loss-of-Function Mutation in MSH5 Abolishes MutSγ Axial Loading and Causes Meiotic Arrest in NOA-Affected Individuals

Non-obstructive azoospermia (NOA), characterized by spermatogenesis failure and the absence of sperm in ejaculation, is the most severe form of male infertility. However, the etiology and pathology between meiosis-associated monogenic alterations and human NOA remain largely unknown. A homozygous MSH5 mutation (c.1126del) was identified from two idiopathic NOA patients in the consanguineous family. This mutation led to the degradation of MSH5 mRNA and abolished chromosome axial localization of MutSγ in spermatocytes from the affected males. Chromosomal spreading analysis of the patient’s meiotic prophase I revealed that the meiosis progression was arrested at a zygotene-like stage with extensive failure of homologous synapsis and DSB repair. Therefore, our study demonstrates that the MSH5 c.1126del could cause meiotic recombination failure and lead to human infertility, improving the genetic diagnosis of NOA clinically. Furthermore, the study of human spermatocytes elucidates the meiosis defects caused by MSH5 variant, and reveals a conserved and indispensable role of MutSγ in human synapsis and meiotic recombination, which have not previously been well-described.

Introduction
To date, about 48.5 million couples worldwide suffer from infertility, 50% of which are attributed to male factors [1]. It has been estimated that about 10% of infertile men are affected by azoospermia, which is the most severe form of male infertility [2]. Azoospermia is classified as obstructive azoospermia (OA) and non-obstructive azoospermia (NOA). By means of testicular biopsy and histological features, the etiology of NOA is classified into Sertoli-cell only, maturation arrest (including spermatocyte or spermatid stage arrest), and hypospermatogenesis [3]. With the widespread use of high-throughput approaches, whole genome sequencing (WGS) and whole exome sequencing (WES) become effective tools for exploring potentially pathogenic NOA-related single gene mutations and a number of genetic mutations were thus identified including MEIOB, SPATA22, ZSWIM7, and C14ORF39 [4][5][6]. However, the causes of the majority of cases of the monogenic alterations in human NOA remain to be explored. 2 of 14 Meiosis specifically occurs in gametogenesis during which the genome halves via two consecutive rounds of cell division. During meiotic prophase, recombination is initiated by the SPO11-generated DNA double-strand breaks (DSBs) [7,8]. Subsequent end resection on DSB produces 3 single-stranded DNA (ssDNA) overhangs [9]. The replication protein A (RPA) complex, consisting of RPA1, RPA2, and RPA3, coats the ssDNA to prevent ssDNA from degradation and the formation of secondary structures [10,11]. Loading of RPA on ssDNA is also a prerequisite for the recruitment of DNA recombinase DMC1 and RAD51 [12]. Later on, DMC1 and RAD51 gradually replace RPA and drive single strand invasion into homologous chromosomes to produce the nascent displacement loop (Dloop) [13][14][15][16][17]. As the D-loop extends, the second end capture takes place and results in the formation of double Holliday junctions (dHJs). Factors such as the MSH4/MSH5 complex and TEX11 are involved in stabilizing both nascent D-loop and dHJ structures [18][19][20]. As a consequence of meiotic recombination, the MLH1/MLH3 complex directs the resolution of dHJs to produce crossover(s) on every pair of homologs that serve as a bond to physically link homologous chromosomes and progressively turn into chiasmata, which facilitates accurate homolog bi-orientation on the spindle [21][22][23][24].
In eukaryotic cells, MSH4 and MSH5 belong to the MutS homolog (MSH) family, forming a heterodimer termed MutSγ, which plays specific roles at multiple steps in meiotic recombination [22,25,26]. In vitro and in vivo evidence revealed that MutSγ recognizes and binds to nascent D-loops and dHJs, stabilizes the recombinant intermediates, promotes crossover formation, and facilitates MLH1/MLH3 to convert these intermediates into mature crossovers [18,27]. Msh5 −/− mice exhibited chromosome synapsis failure and meiotic arrest at a zygotene-like stage, resulting in male and female sterility [28]. Variants in MSH5 are associated with human primary ovarian insufficiency [29] and male infertility [30][31][32].
In the present study, we aimed to investigate the effect of the MSH5 variant on human meiotic recombination, and further revealed the in vivo function of MutSγ in human meiosis. A homozygous MSH5 mutation (c.1126del) was detected from a consanguineous Pakistan family by the WES combined with bio-informatic analyses. Sanger sequencing was conducted to validate that the MSH5 mutation was carried by two idiopathic NOA-affected men in this family. We performed a testicular biopsy for one of the patients harboring the MSH5 mutation, and determined the effect of the MSH5 mutation in the patient's testicular tissues by quantitative real-time PCR. To further elucidate the in vivo function of MutSγ in human meiosis, we conducted immunostaining using meiotic recombination markers on the patient's spermatocyte spread slides.

Clinical Characteristics of NOA-Affected Individuals from a Consanguineous Family
We recruited a consanguineous Pakistan family with two idiopathic NOA-affected men (IV-4 and IV-6) who had no offspring after at least 10 years of marriage ( Figure 1A and Table 1). Physical examinations indicated that they had normal stature, secondary sexual characteristics, and intellectual ability. Chromosome analysis revealed a normal karyotype, and no Y-chromosome microdeletions. Hormone measurements from IV-4 and IV-6 revealed that the levels of LH, prolactin, and testosterone were in the normal ranges, while the FSH level was higher than the reference value (Table 2). Testicular scrotal ultrasound examination reflected the normal testes size, shape, and outlines for IV-4 and IV-6 ( Table 1). Semen analyses were conducted twice following the WHO guidelines and the results showed that both individuals had normal semen volume, but no sperm (Table 1). To further examine their reproductive status, testicular biopsies were performed on IV-6 and a male control with OA whose spermatogenesis was normal. Following H&E staining on the testicular sections, it was revealed that compared to those in the OA control, seminiferous tubules from IV-6 contained spermatogonia and spermatocytes, but were completely devoid of post-meiotic germ cells. These results indicate that the spermatogenesis was arrested at the spermatocyte phase in the patient ( Figure 1B). staining on the testicular sections, it was revealed that compared to those in the OA control, seminiferous tubules from IV-6 contained spermatogonia and spermatocytes, but were completely devoid of post-meiotic germ cells. These results indicate that the spermatogenesis was arrested at the spermatocyte phase in the patient ( Figure 1B).

A Homozygous Frameshift Mutation in MSH5 Is Identified in Two Infertile Individuals
To uncover the potential genetic cause of spermatocyte arrest in this family, WES was performed on all of the available family members including the two infertile individuals (IV-4 and IV-6) and their mother (III-1) ( Figure 1A). The variants detected from the WES data were filtered with a series of criteria ( Figure 2A). In brief, as the individuals were born to a consanguineous family, variants following recessive inheritance pattern in the sequenced family members were considered of priority. Linkage analysis was first performed and five linkage regions were identified ( Figure 2B). The variants within these linkage regions and meeting the following conditions were given preference: (1) variants with minor allele frequency (MAF) <0.01 in the 1000 Genomes, ESP6500, ExAC, and Genome Aggregation Database; (2) variants affecting the protein sequence; (3) loss-of-function variants or potentially deleterious missense variants predicted by software including Sorting Intolerant From Tolerant, PolyPhen-2, and Mutation Taster; and (4) variants in genes expressed in the testis. Consequently, a 1-bp deletion in MSH5 (c.1126del [p.Ser376Ala fs*6] [GenBank: NM_172166.4]) was screened out as a potentially pathogenic variant ( Figure 2A). Homozygosity mapping analysis revealed that the MSH5 variant was within long runs of homozygosity (RoH) regions in the two affected individuals (IV4: RoH = 12 Mb and IV6: RoH = 16 Mb) ( Figure 2C). This MSH5 variant was verified by Sanger sequencing and was confirmed to co-segregate with male infertility in this family ( Figure 1C). The MSH5 variant carried by IV-4 and IV-6 were further confirmed at the mRNA level in peripheral blood or testicular tissues, respectively ( Figure S1). In addition, the 1-bp deletion in MSH5 presumably causes a frameshift that changes the amino acid residue serine to alanine at position 376, which is located in the highly conserved DNA binding domain ( Figures 1E and S2), and introduces a premature translational termination of MSH5 at amino acid position 381 ( Figure 1D). Therefore, we suggest that the identified homozygous MSH5 c.1126del could be pathogenic for male infertility in this pedigree.

Dynamic Localization of MSH5 and MSH4 to Synaptonemal Complexes in Human
To acquire the dynamics of MutSγ in vivo, we analyzed the MSH5 and MSH4 localization patterns in spread spermatocytes from a patient diagnosed with OA by superresolution structured illumination microscopy (SIM). In human spermatocytes, the MSH5 foci first appeared at leptonema, and began to associate with axes on synapsed regions at early-zygonema (45.33 ± 15.99 (SD); 24 nuclei; Figure 3A,C), supposing that MSH5 may function in the early steps of human meiotic recombination. Intensive MSH5 foci were

The Identified MSH5 Mutation Causes mRNA Degradation and Abolishes Axial Localization of MutSγ in the Patient
To evaluate the effects of the identified mutation on MSH5 expression, quantitative real-time PCR was performed and revealed that the level of MSH5 mRNA in the peripheral blood of IV-4 was largely reduced to around 1.17% ± 0.57% (mean ± SD) compared to the control ( Figure 4A). Similarly, only around 2.9% ± 1.8% (mean ± SD) of the MSH5 mRNA expression was detected in the IV-6 testicular tissue compared to the control, indicating that MSH5 mRNA was almost absent from the two affected individuals ( Figure 4B). Further immunofluorescence staining on the spread spermatocytes with antibodies against SYCP3 and MSH5 confirmed a complete lack of MSH5 foci in all spermatocytes from IV-6 ( Figure 4C). To evaluate the effects of the identified mutation on MSH5 expression, quantitative real-time PCR was performed and revealed that the level of MSH5 mRNA in the peripheral blood of IV-4 was largely reduced to around 1.17% ± 0.57% (mean ± SD) compared to the control ( Figure 4A). Similarly, only around 2.9% ± 1.8% (mean ± SD) of the MSH5 mRNA expression was detected in the IV-6 testicular tissue compared to the control, indicating that MSH5 mRNA was almost absent from the two affected individuals ( Figure  4B). Further immunofluorescence staining on the spread spermatocytes with antibodies against SYCP3 and MSH5 confirmed a complete lack of MSH5 foci in all spermatocytes from IV-6 ( Figure 4C).  MSH5 contains two regions (152-263 aa and 748-834 aa) that specifically interact with MSH4 to form the MutSγ heterodimer during meiosis [33][34][35]. To reveal the effect of the MSH5 variant on MSH4 localization, we performed immunofluorescence staining using antibodies against SYCP3 and MSH4 on the human spread spermatocytes. Unlike the control spermatocytes in which the axes were decorated with abundant MSH4 foci, MSH4 signals were absent in all spermatocytes from IV-6 ( Figure 4D). These results revealed that the homozygous MSH5 c.1126del mutation is deleterious, provoking MSH5 mRNA degradation and abolishing the axial binding of MutSγ in the patient's spermatocytes.

Meiotic Arrest at Zygotene-like Stage with Synaptic Defects in the Patient
To analyze the meiotic progression in more detail, immunofluorescent staining of SYCP3 and SIX6OS1, a central element of the synaptonemal complex, was conducted. In the control spermatocytes, three sub-stages of meiotic prophase I, leptotene, zygotene, and pachytene were identified, and the pachytene spermatocytes constituted approximately 63% of the total populations. Strikingly, no pachytene cells were detected in the patient's samples, instead, over 80% of cells were zygotene (and-like) cells, which were significantly higher than the frequency of zygotene cells in the control samples ( Figure 5B). Based on the extent of synapsis, two deviant types of zygotene (and-like) cells in the patient could be distinguished: (I) Type I cells with linear SIX6OS1 signals detected along paired chromosome axes, indicating partial synapsis; and (II) type II cells had linear lateral elements but were completely devoid of the central element, indicating the absence of synapsis; these cells were termed as zygotene-like and accounted for 32.62% of the total spermatocytes in the patient ( Figure 5A,B). These results collectively indicate that the patient harboring MSH5 c.1126del had meiosis arrest at a zygotene-like stage, accompanied with severe synapsis failure between homologous chromosomes.

Failure of Meiotic DSB Repair in Spermatocytes from the Patient
MutSγ is known to bind and stabilize the recombinant intermediates to promote recombination-based DSB repair [18,27,36]. To investigate meiotic DSB repair after the disruption of MutSγ, we performed immunofluorescence staining on the patient's spread spermatocytes for SYCP3 and γH2AX, a marker of DNA breaks. Cloud-like γH2AX signals were distributed on some chromosome pairs in zygotene spermatocytes from the control. While in the patient, the overall intensity of γH2AX signals in the zygotene (and-like) cells were obviously higher than in the control zygotene cells. In the type I zygotene cells, strong γH2AX signals covered most of the unsynapsed homologs. In zygotene-like spermatocytes of the patient, the γH2AX signals were dispersed on every chromosome and appeared to be stronger than those in the type I zygotene cells ( Figure 5C). These results demonstrate that the DSBs failed to be repaired in the patient's spermatocytes.

Discussion
In this study, we identified a homozygous MSH5 c.1126del in two NOA individuals from a consanguineous family, which triggered degradation of MSH5 mRNA and loss of MSH5-MSH4 localization onto chromosome axes in the patients' spermatocytes. A series of cytological studies in human spermatocytes revealed that the failure of MSH5-MSH4 localization on the chromosome axes impeded synapsis and meiotic DSB repair, resulting in meiotic arrest and azoospermia. Thus, our findings revealed that a novel biallelic variant of MSH5 (c.1126del) is associated with human NOA.
MutSγ is well-known for its role in meiotic recombination in multiple organisms [28,[37][38][39][40][41]. Previous studies on mouse models illustrated that loss of either Msh5 or Msh4 resulted in meiotic arrest at a zygotene-like stage, with aberrant pairing, insufficient synapsis, and a failure to complete DSBs repair [28,38,42]. Up to now, several variants in MSH4 and MSH5 have been reported in infertile men and women. However, the cytological consequence of MutSγ disruption in humans remains uncharacterized. Here, we showed that the lossof-function mutation in MSH5 caused meiotic defects similar to that in mice including the zygotene-like arrest and defects in synapsis and DSB repair. However, the extent of synapsis defects is different between the mouse and human mutants. In our patient, 32.62% spermatocytes formed the full (or almost full) length of the lateral axes but completely lacked central element signals, which was not described in the Msh5 knockout mice [28]. It thus seems that MSH5 may serve a more important role in homolog synapsis in humans than in mice.
In mice, the degree of chromosome pairing and synapsis in Msh4 −/− /Msh5 −/− mice is more similar to that in Msh5 −/− mice, and is less advanced than in Msh4 −/− mice [38]. Moreover, MSH4 foci on chromosome cores were dramatically decreased and became fainter in mutants with the disruption of the MSH5 ATP binding domain [43], suggesting that the MSH5 acts epistatic to MSH4 during mouse meiosis. Notably, in our patient, MSH5 expression was almost completely abolished, and we did not detect any MSH4 foci on the chromosome axes in all spermatocytes either, indicating that MSH4 recruitment or the association of MSH4 to the axes is dependent on MSH5. Referring to the mouse studies, we can infer that MSH5 is also likely to be a prerequisite for MSH4 recruitment and retention on the axes in human spermatocytes, and that the infertility outcome in the patient carrying the MSH5 mutation (c.1126del) is supposed to be caused by the abolished axial localization of both MSH4 and MSH5.
Recently, several pathogenic variants in MSH5 have been found in NOA individuals [30,32]. Our study provides further supporting evidence on the pathogenicity of MSH5 mutation in male infertility, improving the future genetic diagnosis of infertile individuals in clinic. More importantly, based on studies of human spermatocytes, we elucidate the meiosis defects caused by the MSH5 variant and demonstrate a conserved and indispensable role of MutSγ in human synapsis and meiotic recombination, which have not been well-described previously.

Clinical Samples
A consanguineous Pakistani family was enrolled in this study. Written informed consent was received from all participants prior to the onset of the study. Semen analyses were performed according to the WHO guidelines.

Hormone Measurement
Before the blood was collected for hormone analysis, patients were advised to stop certain medications, which included not taking drugs such as oral steroids or estrogen supplements, TSH and testosterone level elevators, etc. The patient's blood was collected at fasting condition (overnight) to avoid possible food interventions that can raise certain hormone levels in the blood. For hormone analysis, peripheral blood samples were collected from the available individuals using 5cc disposable sterile syringes (Becton Dickinson supplies Pakistan) and adopting standard protocols (WHO 2010). A total of 3 mL of peripheral blood was added to the serum separation tubes (SSTs) in the Tube with Gel and Clot Activator (VITREX Medical, Herlev, Denmark) for serum separation. Blood was kept in the SSTs and set for 30 min to 1 h at room temperature to clot before spinning and separating. This was then centrifuged for 5 min at approximately 1000× g and a clean pipette was used to remove the separated serum and immediately loaded to the COBAS e 411(Roche, Basel, Switzerland) special chemistry analyzer for hormone analysis.

Pre-Operation Preparation of Patient
The patient was evaluated clinically for the procedure and anesthesia. The necessary hormone analyses, complete blood count (CBC), viral marker tests (HIV, HCV, HBS), and ultrasonographic testicular scans were performed. To control the hospital acquired infection, the required intravenous fluids and antibiotics were administered, the patient was kept nothing by mouth (NBM), and no foods and fluids were taken to keep the stomach empty.

General Anesthesia (GA)
Before GA, the patient remained NBM for at least 10 h and was thoroughly evaluated clinically including weight, heart ECG, BP, and age factors.

Surgical Removal of Testicular Tissue
The skin over the testicle was cleaned with a germ-killing (antiseptic) medicine. The area around it was covered with a sterile towel. A small surgical scrotal incision of 2-3 cm was made for the exposure of the tunica albuginea of the testis. A small incision in the capsule of the testicular of about 0.5 cm was made to obtain a biopsy of about 3 × 3 × 3 mm. For the adequate classification of spermatogenesis, while removing the tissue, it was ensured that it should contain at least 100 seminiferous tubules. At the same time, the tissue was not squeezed with forceps to not to disrupt the testicular tissue architecture and hamper the proper evaluation of the seminiferous tubules. The opening in the testicle was closed with a stich.

Whole-Exome Sequencing and Variant Filtration
Total genomic DNA was isolated from the peripheral blood of the study samples. Whole-exome capture and sequencing were performed using the AlExome Enrichment Kit V1 (iGeneTech, Beijing, China) and Hiseq2000 platform (Illumina, San Diego, CA, USA) following the standard procedures. The reads were aligned to the human genome reference assembly (hg19) using a Burrows-Wheeler Aligner with default parameters. Then, the Picard software (https://sourceforge.net/projects/picard/, download date: 24 September 2015) was employed to remove polymerase chain reaction duplicates. DNA sequence variants were called using the Genome Analysis Toolkit HaplotypeCaller (http: //www.broadinstitute.org/gatk/, download date: 30 June 2018). Variants were filtered as previously described [44,45].

RNA Extraction, Quantitative Polymerase Chain Reaction (qPCR) and Reverse Transcription-Polymerase Chain Reaction (RT-PCR)
Human testicular tissue or peripheral blood were ground and solubilized in 1 mL TRIzol (Accurate Biology AG21101, Hunan, China). The total RNA was extracted according to the manufacturer's protocol, as previously described [44], with minor modifications made as follows. A total of 200 µL chloroform was added to 1 mL TRIzol and centrifuged at 12,000× g for 15 min at 4 • C. After centrifugation, the upper clear aqueous phase was transferred to a 1.5 mL centrifuge tube, then 1 mL isopropanol was added and centrifuged at 12,000× g for 10 min at 4 • C. Discarding the supernatant, 1 mL of 75% ethanol was then added and centrifuged at 12,000× g for 10 min, and the obtained total RNAs were dissolved by RNase-free water. After denatured by heating at 56 • C in a metal bath for 5 min, the cD-NAs were synthesized with the Prime Script RT Reagent Kit (TaKaRa RR047A, Shiga, Japan) according to the manufacturer's protocols. The concentration and purity of cDNA were measured by NanoDrop 1000 Spectrophotometer (Thermo Fisher Scientific corporation, Waltham, MA, USA). The qPCR was performed with TransStart Top Green qPCR Super Mix (TransGen Biotech AQ132, Beijing, China) and a Step One Real-Time PCR System (Applied Biosystems, Thermo Fisher Scientific corporation, Waltham, MA, USA). We calculated the relative mRNA levels by normalizing to ACTB (MIM: 102630). The relative mRNA levels in the control were regarded as 1.0, and the fold change in the affected individual was compared to the control. Primer sequences are listed in Supplementary Table S1. RT-PCR was conducted using templates from the peripheral blood or testicular cDNA, respectively. For the cDNA obtained from the peripheral blood of the male control and individual IV-4, PCR reactions were performed as follows: first round of PCR, 5 min at 94 • C, followed by 38 cycles of 15 s at 98 • C, 30 s at 58 • C, and 30 s at 72 • C; second round of PCR, 5 min at 94 • C, followed by 36 cycles of 15 s at 98 • C, 30 s at 55 • C, and 30 s at 72 • C. For the cDNA obtained from the testicular tissues of the male control and individual IV-6, PCR reactions were performed as follows: 3 min at 95 • C, followed by 38 cycles of 15 s at 95 • C, 15 s at 58 • C, and 30 s at 72 • C. The RT-PCR products were electrophoresed on a 2% agarose gel, followed by Sanger sequencing. Primer sequences are listed in Supplementary Table S1.

Hematoxylin and Eosin Staining
Human testicular tissues were fixed in Bouin's solution overnight, then embedded into the paraffin, and sectioned at a 5 µm thickness. The H&E staining was conducted according to previously described protocols [6]. Briefly, the tissue slides were deparaffinized in xylene for 20 min, then rehydrated with gradient ethanol: 100%, 90%, 80%, and 50% (5 min for each concentration) and sequentially stained with hematoxylin for 5 min and eosin for 10 s. After stepwise dehydration with 50%, 80%, 90%, and 100% ethanol (2 min for each concentration) and transparency in xylene for 5 min, the tissue sections were sealed with neutral resin. The images were captured via a Nikon ECLIPSE 80i microscope (Nikon Instruments, Tokyo, Japan) with a DR-Ri1 camera and processed with NIS-elements Basic Research software (Nikon Instruments, Tokyo, Japan).

Spermatocyte Nuclear Surface Spreading and Immunofluorescence Staining
The surface-spreads of the human spermatocytes were prepared according to the previously described protocols [46]. Immunofluorescence staining was conducted according to the previously described protocols [6,47]. Briefly, slides were blocked with 3% non-fat milk in phosphate-buffered saline (PBS) for 10 min and then incubated with primary antibodies (mixed with 3% non-fat milk) overnight at 37 • C in a humidified chamber. After three washes in PBS containing 0.35% Triton X-100 (PBST), secondary antibodies (mixed with 3% non-fat milk) were applied for 1 h at 37 • C. Subsequently, the slides were washed three times in PBST and mounted with the Vectashield Medium (Vector Laboratories H1000, Newark, CA, USA). Images were captured with an Olympus BX61 microscope (Tokyo, Japan) with a CCD camera (QICAM Fast 1394, QImaging, Burnaby, BC, Canada) and processed with Image-Pro Plus software (Media Cybernetic, Rockville, MD, USA). Super-resolution imaging microscopy analysis was performed using a Nikon NSIM super-resolution microscope system and NIS-Elements 2 image processing software (Nikon Instruments, Tokyo, Japan). The antibodies are listed in Supplementary Table S2.

Statistical Analysis
GraphPad Prism Software (GraphPad Software, San Diego, CA, USA) was used to perform the statistical analysis. Statistical results were presented as mean ± SD in Figures 3C,D and 4A,B. The Student's t-test was used for all statistical analyses. The difference was considered as significant when the p values were <0.05.


Simultaneous metabolite MALDI-MSI, whole exome and transcriptome analysis from formalin-fixed paraffin-embedded tissue sections

Matrix-assisted laser desorption ionization mass spectrometry imaging (MALDI-MSI) allows spatial analysis of proteins, metabolites, or small molecules from tissue sections. Here, we present the simultaneous generation and analysis of MALDI-MSI, whole-exome sequencing (WES), and RNA-sequencing data from the same formalin-fixed paraffin-embedded (FFPE) tissue sections. Genomic DNA and total RNA were extracted from (i) untreated, (ii) hematoxylin-eosin (HE) stained, and (iii) MALDI-MSI-analyzed FFPE tissue sections from three head and neck squamous cell carcinomas. MALDI-MSI data were generated by a time-of-flight analyzer prior to preprocessing and visualization. WES data were generated using a low-input protocol followed by detection of single-nucleotide variants (SNVs), tumor mutational burden, and mutational signatures. The transcriptome was determined using 3’-RNA sequencing and was examined for similarities and differences between processing stages. All data met the commonly accepted quality criteria. Besides SNVs commonly identified between differently processed tissues, FFPE-typical artifactual variants were detected. Tumor mutational burden was in the same range for tissues from the same patient and mutational signatures were highly overlapping. Transcriptome profiles showed high levels of correlation. Our data demonstrate that simultaneous molecular profiling of MALDI-MSI-processed FFPE tissue sections at the transcriptome and exome levels is feasible and reliable.

INTRODUCTION
In order to investigate the molecular makeup of tissues in an integrative fashion, multilevel "omics"-studies have gained great relevance [1][2][3][4] . Although desirable, it is challenging to assess multiple omics levels from the same tissue sample. However, with the advancement of omics methodology, it is now possible to generate high-quality genomic and transcriptomic profiles from small numbers of cells and nucleic acids of limited quality 5,6 . This paved the ground for performing different omics measurements from the same tissue specimen. Here, we present the data of matrix-assisted laser desorption ionization mass spectrometry imaging (MALDI-MSI), whole-exome sequencing (WES), and RNA sequencing (RNA-seq) generated from the same formalin-fixed paraffin-embedded (FFPE) tissue sections and demonstrate the feasibility and robustness of the applied protocols.
In order to investigate proteomics and metabolomics in an untargeted manner, mass spectrometry, which determines the mass-to-charge (m/z) ratio of ionized particles, is the method of choice 7,8 . MSI even extends the analytical capabilities of mass spectrometry by visualizing the spatial distribution of individual molecules within their histological context. MSI, which primarily is used to capture protein and metabolite levels, has been recently combined with approaches measuring additional molecular layers. Kazdal et al. described a multiplex approach that applied MSI with digital PCR to the same tissue sections 9 . The possibility of combining metabolic and genetic information from FFPE tissues was also investigated by combining MSI with fluorescence in situ hybridization 10 . MALDI-MSI can also be used to discriminate molecularly homogeneous regions within tissue sections prior to manual or laser microdissection for further molecular analyses 11 . To investigate clinical tissue samples at the genomic and transcriptomic levels, next-generation sequencing (NGS) has become state of the art and the simultaneous applicability of MALDI-MSI and NGS methodology on FFPE tissue sections would be highly desirable 4,12 .
Combining omics approaches has been done in numerous studies that integrated genomics, transcriptomics, proteomics, and metabolomics data generated from clinical samples 2,13 . However, so far, the feasibility of the generation of WES, RNA-seq, and MALDI-MSI data from the same FFPE tissue section has not been demonstrated.
In this technical note, we present a sequential coupling of MALDI-MSI with WES and RNA-seq on the same FFPE tissue section for three different specimens of head and neck squamous cell carcinoma (HNSCC).

MATERIAL AND METHODS Tumor tissue specimens
Tissue specimens of histologically confirmed HNSCC from three patients who had undergone surgical resection were provided in the framework of the Clinical Cooperation Group "Personalized Radiotherapy in Head and Neck Cancer" (Ludwig-Maximilians-University of Munich and Helmholtz Zentrum München). According to common pathology practice, the tissues were fixed with formalin for 24 h prior paraffin embedding. FFPE tissue sections (3 µm) were prepared using a microtome (HM340E, Thermo Scientific, Waltham, Massachusetts, USA) and mounted on indium tin oxide-treated poly-L-lysine-coated glass slides.

MALDI-MSI
Three experimental setups for testing the effect of deparaffinization, hematoxylin and eosin staining, and MSI on RNA-seq and WES data were used (see Fig. 1). In the first, referred to as FFPE, the FFPE sections were deparaffinized by incubation for 60 min at 60°C prior paraffin removal by two incubation steps in xylene for 7 min each. In the second, referred to as FFPE-HE, sections were deparaffinized prior to hematoxylin and eosin staining using a standard protocol 14 . In the third, referred to as FFPE-MSI, deparaffinized and HE-stained tissue sections were subjected to the MSI workflow. For this purpose, the deparaffinized section first was coated with a 9-aminoacridine (9-AA) ionization matrix. The matrix was prepared by dissolving 10 mg/ml 9-AA (Sigma-Aldrich, St. Louis, Missouri, USA) in 70% methanol. The tissue coating was performed using an automated spray system (Micro Fraction Collector, MALDI-Spotter SunCollect, SunChrom, Friedrichsdorf, Germany). Subsequently, mass spectrometric data were measured with an Ultraflex III MALDI-TOF instrument (Bruker Daltonics, Bremen, Germany) in linear negative mode, detecting metabolites mainly in the mass range of 100-1060 Da and a lateral resolution of 60 µm. The tissue sections were stained with hematoxylin and eosin, scanned with a digital slide scanning system (Mirax Desk, Carl Zeiss MicroImaging, Göttingen, Germany), and co-registered with the MALDI-MSI measurement in the FlexImaging software (Bruker Daltonics, Bremen, Germany), to match the MS data with the histological features of the tissue sections. Metabolites likely to be reflected by the m/z species were identified by requesting the Human Metabolome Database (HMDB) with an allowed tolerance of 10 ppm.

Next-generation sequencing
Genomic DNA and total RNA were isolated and purified simultaneously from the whole tissue sections using the AllPrep DNA/RNA FFPE Kit according to the manufacturer's protocol (Qiagen, Hilden, Germany), followed by assessing nucleic acid integrity with the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, California, USA) and the Agilent DNA 12000 Assay Kit or the Agilent RNA 6000 Pico Assay Kit, respectively.
Whole-exome sequencing. In order to determine the fragmentation status and further amplification potential of the extracted gDNA, the TruSeq FFPE DNA Library Prep QC Kit (Illumina, San Diego, California, USA) was used according to the manufacturer's protocol and executed on a ViiA 7 Real-Time PCR System (Thermo Scientific, Waltham, Massachusetts, USA). This quality assessment approach is based on a genomic qRT-PCR, where the Ct values (cycle thresholds) of the tissue sample DNAs are set in relation to the Ct of a validated amplicon control DNA (proprietary). Depending on the obtained ratio, the samples were classified into different quality levels which determine the input quantity for the DNA library preparation. The SureSelect XT v6 (target size: 60 Mb) Target Enrichment System (Agilent Technologies, Santa Clara, California, USA) was used to construct a whole exome library which was subsequently sequenced on a HighSeq4000 system (Illumina, San Diego, California, USA) according to the manufacturer's protocol.
3' mRNA sequencing. Total RNA integrity was assessed by means of the DV200 value (i.e., the percentage of fragments >200 nucleotides) using an Agilent RNA 6000 Pico Chip Kit on the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, California, USA). 3' mRNA sequencing libraries were generated using the QuantSeq 3' mRNA-Seq Library Prep Kit (Lexogen, Vienna, Austria) according to the manufacturer's instructions. For library amplification, PCR cycles were determined using the PCR Addon Kit for Illumina (Lexogen, Vienna, Austria). Before sequencing on a HighSeq4000 system (Illumina, San Diego, California, USA), quantity and quality of sequencing libraries were assessed using the Quanti-iT PicoGreen dsDNA Assay Kit (Thermo Scientific, Waltham, Massachusetts, USA) and the Bioanalyzer High Sensitivity DNA Analysis Kit (Agilent Technologies, Santa Clara, California, USA).

Bioinformatic analyses
Single-nucleotide variants and genomic copy number changes. The WES profiles were preprocessed and analyzed according to the GATK best practices workflow "Somatic short variant discovery" (https:// gatk.broadinstitute.org/hc/en-us/articles/360035894731-Somatic-shortvariant-discovery-SNVs-Indels-). Implementation of the code was mainly adapted from the GATK workflows GitHub repository (https://github.com/ gatk-workflows). Before applying the gatk4-data-processing workflow, the raw fastq files from different sequencing lanes were concatenated using the "cat" bash command. The unaligned bam files were then subjected to alignment using bwa-mem, Mutect2 calling of SNVs, and indels. GATK4-Mutect2 was run in tumor-only mode. SNVs which were quality-flagged by Mutect2 were filtered out. The resulting vcf files were imported into R and further processed using functions of the maftools R-Bioconductor package 15 . In addition, only SNVs were kept that were covered by more than 10 reads. Furthermore, the variants had to be present as coding or Fig. 1 Workflow for RNA and exome sequencing on MALDI-MSIprocessed FFPE tissue sections. For examinations of the effect of the MSI workflow on the genome and transcriptome, the FFPE samples were sectioned and subsequently processed in three different approaches. As a control approach, one sample was extracted directly after dissection (FFPE) and one sample was dissected and stained with hematoxylin and eosin (FFPE-HE). In the discovery approach, MSI with an additional HE staining was performed to follow the regular procedure of an MSI experiment (FFPE-MSI). After scanning the stained tissue for the MSI procedure, the same tissue was used for the extraction of gDNA and total RNA. Then the integrity of the nucleic acids was examined, the libraries were prepared and the samples were sequenced accordingly.
noncoding variant in the Cosmic (version 92) database and, furthermore, their population allele frequency (any ethnicity) was not allowed to exceed 10 -5 according to the gnomAD database (https://gnomad. broadinstitute.org). Furthermore, CNVs of the top 100 frequently mutated genes were excluded 16 . The tumor mutational burden (TMB) was calculated as the ratio between the number of called variants per megabase. For calling mutational signatures with functions of the deconstructSigs R package, the Cosmic SBS signatures v3 (https://cancer. sanger.ac.uk/cosmic/signatures) were used.
Analysis of RNA-sequencing data. The RNA-seq data were preprocessed as follows: the forward-reads of the 150 bp paired-end sequencing were subjected to adapter trimming using BBDuk (https://jgi.doe.gov/data-andtools/bbtools/bb-tools-user-guide/bbduk-guide/), followed by alignment to human genome 38 (hg38) using the STAR aligner. Aligned reads were counted by htseq-count (https://htseq.readthedocs.io/en/release_0.11.1/ count.html). The count files were then imported into R for further processing using the R DESeq2 package 17 . The counts per Ensembl transcript id were summarized per gene and the names were converted to HGNC gene symbols. Only genes with a total average of 10 counts per profile were kept in the dataset. For pairwise-correlation (Spearman) and visualization by scatter plots the profiles were vst-transformed.
Quality assessment of sequencing data. For both, WES and RNA-seq, quality assessment of raw and mapped reads was performed using FastQC and the results were summarized and visualized using MultiQC.

Matrix-associated add-on experiment
In order to assess the potential impact of the matrix on the results of the sequencing approaches, additional matrix-based analyses were performed. For this purpose, an approach without prior matrix application, an approach with prior matrix application, and an approach in which the process of washing off the matrix after the MSI run was simulated were carried out (see Supplementary Fig. S1). A 9-AA matrix was used, which was sprayed on as described above. Furthermore, in order to estimate the minimum amount of sample material required for successful amplification of the RNA and subsequent sequencing, a sample consisting of 1/2, 1/4, or 1/8 tissue was included from the approach with removed matrix in addition to the complete tissue isolate. Extraction of total RNA was performed with the RNeasy FFPE Kit (Qiagen GmbH, Hilden, Deutschland) with slight modifications. To increase the digestion activity of proteinase K on contaminating proteins and endogenous nucleases, samples were incubated overnight at 56°C. To remove all residues of salts, the RPE buffer washing volume was increased to 500 µl in two successive washing steps. For analysis of nucleic acid integrity, reverse transcription was performed on the RNA. The SuperScript VILO cDNA synthesis Kit (Thermo Scientific, Waltham, Massachusetts, USA) was applied according to the manufacturer's protocol and subjected to qRT-PCR (ViiA 7 Real-Time PCR System, Life Technologies, Carlsbad, California, USA) using TaqMan gene expression assays (Thermo Scientific, Waltham, Massachusetts, USA) against the housekeeping genes actin beta (ACTB, assay ID: Hs01060665_g1, amplicon length: 63 bp), glyceraldehyde-3-phosphate dehydrogenase (assay ID: Hs99999905_m1, amplicon length: 122 bp) and phosphoglycerate kinase 1 (PGK1, assay ID: Hs99999906_m1, amplicon length: 75 bp). Assessment of RNA integrity and 3' mRNA sequencing was performed as described above.

RESULTS AND DISCUSSION
The coupling of omics methods that capture different molecular layers from the same biological sample is the prerequisite of integrative multilevel integration approaches. Here, we aimed to test the feasibility of determining the spatially resolved metabolite level using MALDI-MSI, the genome level using WES, and the transcriptome level using RNA-seq from the same FFPE tissue sections. For each molecular level, the quantity, quality, and plausibility of the resulting data were evaluated and compared for three HNSCC FFPE tissue samples, each of which was FFPE, FFPE-HE, and FFPE-MSI processed (Fig. 1).

MALDI-MSI at the metabolite level
The MSI measurement at a local resolution of 60 µm resulted in a total number of 89,685 spectra. All spectra were detected in a mass range between m/z 100 and 1060 to include the common metabolic masses 18 . The calibration was performed using phosphorus red. The overall spectra were comparable with regard to mass signals. Sample 1 contained 347, sample 2 contained 344, and sample 3 contained 377 mass signals after peak picking, based on a signal-to-noise ratio of 2. As expected, the peak of mass m/z 193.092, which corresponds to the molecular weight of the matrix substance 9-AA, formed the maximum of all components present in the spectrum with an intensity of 83.69. An HMDB database request with the identified m/z species revealed 772 metabolites for tissue sample 1, 1314 metabolites for sample 2, and 990 metabolites for sample 3. The three tissue samples shared 296 database-revealed metabolites (SI File 1). To ensure plausibility of the MSI measurement, metabolites with specific abundances in the tumor (m/z 152.9452) and stroma (m/z 148.8547), respectively, were visualized (Fig. 2). The histological composition of the tumor and tumor stroma was well reflected by these two m/z species. This, together with the quantitative QA measures demonstrates the plausibility of the MALDI-MSI data generated. Quality of genomic DNA Following the qRT-PCR run, baseline correction was performed on the generated data and the average Ct value of the amplicon control DNA (ACD1) was subtracted from the Ct value of the samples. This corrected ΔCt value was used to classify the samples into the following different quality levels: ΔCt value between -1 and 1: "high quality", recommended input quantity: 10 ng; ΔCt value between 1.0 and 2.5: "medium quality", recommended input quantity: 20-50 ng; ΔCt value between 2.5 and 4.0: "low quality", recommended input quantity: 50-100 ng. The input quantity for DNA library preparation was adjusted according to this classification. Tissue 1 had mean ΔCTs

Quality of total RNA
The quality of the RNA was assessed by evaluating the DV200 values. According to the percentage of fragments with a size greater than 200 nucleotides, the quality of RNA integrity is classified as high (>70%), medium (50-70%), or low (30-50%) quality or rather too degraded (<30%), respectively. Accordingly, the FFPE (15%), FFPE-HE (18%), and FFPE-MSI (19%) samples of tissue 1 were classified as too degraded. Tissue 2 with DV200 values just below 50 (FFPE: 48%, FFPE-HE: 49%, FFPE-MSI: 48%) is in the low-quality range and tissue 3 with values (FFPE: 31%, FFPE-HE: 27%, FFPE-MSI: 26%) around 30 is between low RNA quality and too degraded samples. The RNA integrity of the samples is in a lower range, which was to be expected based on the empirical values for RNA with FFPE processing. However, since all samples had fragments in the range >200 nucleotides and the manufacturer's quality assessment only addressed hypothetical chances of success, all samples were subjected to RNA-seq library preparation.
The TMB, reflecting the percentage of the genome covered by the whole exome analysis that is affected by SNVs, was 4.4% on average for the treatments of tissue  When comparing the similarity of the expression profiles using Spearman correlation all pairwise comparisons between the differently treated samples of the samples showed correlation coefficients greater than 91% (Fig. 3D) In hierarchical clustering analysis of the Euclidean distance of the log-transformed (variance stabilized transformation) expression values, all samples belonging to the same tissue clustered together (Fig. 3E).

RNA-sequencing raw and processed data quality
Hierarchical clustering of the distance matrix (Euclidean) from log-transformed expression values, which were built from read counts per gene by variance transformed transformation, (vst) showed co-clustering of expression profiles from the same tissues. The expression of the X-inactive specific transcript gene was consistent with the sex of the patients, which, together with the overall high correlation between profiles from the same tissues and the low correlation between patients, demonstrates the good feasibility and plausibility of using 3'-RNA-seq.
In conclusion, we show that method coupling of MALDI-MSI, WES, and RNA-seq is feasible and can generate technically sound and plausible data. As typical for whole exome data generated from FFPE tissues in tumor-only mode, a high proportion of technically artificial variants were detected. This limitation could be addressed by including matched normal tissue samples for more accurate detection of SNVs.


s2fieldsofstudy: Biology Medicine

text:
Two New Potential Therapeutic Approaches in Radiation Cystitis Derived from Mesenchymal Stem Cells: Extracellular Vesicles and Conditioned Medium

Simple Summary Pelvic radiotherapy is a major therapeutic weapon in the management of pelvic tumors. Despite improvements in radiation techniques, pelvic irradiation is responsible for symptoms that impact the quality of life of our patients. Thus, the incidence of radiation cystitis remains stable over time and the therapeutic possibilities remain limited. Extracellular vesicles (MSC-EVs) or conditioned medium from human mesenchymal stromal cells (MSC-CM) have demonstrated therapeutic potential by promoting tissue repair. We have evaluated their efficacy in preventing fibrosis in a model of radiation cystitis in vitro. Abstract Background: Radiation cystitis (RC) results from chronic inflammation, fibrosis, and vascular damage. The urinary symptoms it causes have a serious impact on patients’ quality of life. Despite the improvement in irradiation techniques, the incidence of radiation cystitis remains stable over time, and the therapeutic possibilities remain limited. Mesenchymal stem/stromal cells (MSC) appear to offer2 a promising therapeutic approach by promoting tissue repair through their paracrine action via extracellular vesicles (MSC-EVs) or conditioned medium from human mesenchymal stromal cells (MSC-CM). We assess the therapeutic potential of MSC-EVs or MSC-CM in an in vitro model of RC. Methods: in vitro RC was induced by irradiation of human bladder fibroblasts (HUBF) with the small-animal radiation research platform (SARRP). HUBF were induced towards an RC phenotype after 3 × 3.5 Gy irradiation in the presence of either MSC-EVs or MSC-CM, to assess their effect on fibrosis, angiogenesis, and inflammatory markers. Results: Our data revealed in vitro a higher therapeutic potential of MSC-EVs and MSC-CM in prevention of RC. This was confirmed by down-regulation of α-SMA and CTGF transcription, and the induction of the secretion of anti-fibrotic cytokines, such as IFNγ, IL10 and IL27 and the decrease in the secretion of pro-fibrotic cytokines, IGFBP2, IL1β, IL6, IL18, PDGF, TNFα, and HGF, by irradiated HUBFs, conditioned with MSC-EVs or MSC-CM. The secretome of MSC (MSC-CM) or its subsecretome (MSC-EVs) are proangiogenic, with the ability to induce vessels from HUVEC cells, ensuring the management of bladder vascular lesions induced by irradiation. Conclusion: MSC-EVs and MSC-CM appear to have promising therapeutic potential in the prevention of RC in vitro, by targeting the three main stages of RC: fibrosis, inflammation and vascular damage.

Introduction
External pelvic radiation therapy is an important tool in the treatment modality for pelvic cancers such as prostate cancer. Irradiation techniques have improved over time, such as intensity-modulated radiation therapy (IMRT), stereotactic radiation therapy, and image-guided brachytherapy. These techniques allow us to deliver increasingly effective doses in smaller volumes while significantly improving treatment tolerance [1].
However, the bladder is a critical organ sensitive to low-dose radiation. Despite improvements in technology, pelvic irradiation remains a cause of acute and/or late adverse events affecting the bladder. The term "radiation cystitis" includes all damage and symptoms of the bladder following radiation to the pelvic organs.
Severity is related to radiation exposure, total dose administered, and schedule of administration and fractionation. Patients with hypertension, diabetes mellitus, history of abdominal surgery, and concurrent chemotherapy have been reported to have a higher risk of radiation cystitis, especially in advanced patients [2]. Manea et al. suggested that bladder neck, after high dose exposures (such as after brachytherapy treatment) may be at higher risk of [3].
Acute radiation cystitis was defined as any adverse event that occurred during or within three months of the end of radiation therapy. Clinical symptoms may include pollakiuria, cystalgia, increased frequency and frequency of urination during the day and night (polyuria), dysuria, and increased urinary urgency. Late radiation cystitis is defined as a pelvic radiation-related adverse event that occurs at least three months and possibly years after completion of radiation therapy. The most typical clinical feature is repeated hematuria of varying severity.These adverse events can affect the patient's quality of life. The clinical management of storage symptoms for acute and late radiation cystitis is largely symptomatic with analgesics, anti-inflammatory drugs, intravesical instillations, hyperbaric oxygen therapy (HBOT) and can go up to cystectomy and urinary diversion. Good hydration is recommended for patients in order to increase diuresis, cleanse the bladder, and avoid urinary obstruction resulting from blood clots [4].
RC damage includes vascular lesions, fibrosis and inflammation. Human bladder fibroblasts (HUBFs) are the key cell in this adverse event.
Mesenchymal stem cells/stromal cells (MSC) are a promising alternative therapy for the treatment of fibrosis due to their proangiogenic potential, immunomodulatory effects, and promotion of tissue repair [5,6]. However, MSC present many challenges, such as their variability, scalability and deployment, limit their clinical applications [7].
The beneficial effects of MSCs can be attributed to their paracrine effects. This can be achieved by regulating its environment by secreting specific components (MSC-CM) and including extracellular vesicles (MSC-EV) [8].
The beneficial effects of MSCs may be attributed to their paracrine action. This may be achieved through the conditioning of its environment by secretion of specific components (MSC-CM) and among them extracellular vesicles (MSC-EVs) [8].
In fact, MSC-EVs, including exosomes and microvesicles, have shown remarkable therapeutic properties in many pathophysiological conditions, with the potential to repair various damaged or diseased tissues and organs, such as nervous system, heart, lung, kidney, bone marrow or cartilage, among others [9,10]. Likewise, MSC-CM has shown therapeutic potential in various fields such as myocardial infarction, stroke and acute and chronic ischemia, neurodegenerative diseases, spinal cord injury, alopecia, acute and chronic wounds, acute liver injury and failure, lung injury, periodontal tissue injury, male infertility and soft tissue and bone defects [11,12].
Thus, MSC-EVs and MSC-CM appear to be promising therapeutic candidates to slow the development of radiation fibrosis and maintain tissue integrity in the long term.
To date, few studies have evaluated their antifibrotic, proangiogenic and immunomodulatory potential, the three key steps in the management of radiation cystitis.
In our study, we assess the therapeutic potential of MSC-EVs and MSC-CM in a human in vitro model of RC.
We conducted a comparative study on the ability of MSC-EVs and MSC-CM in the regulation of gene transcription, modulation of the secretion of pro-and anti-angiogenic proteins and the secretion of pro-and anti-inflammatory cytokines in human bladder fibroblasts (HUBFs).
Our results demonstrated the therapeutic potential of MSC-EVs and MSC-CM in the prevention of RC, as confirmed by down-regulation of α-SMA, CTGF and Col1α2 transcription and up-regulation of antifibrotic genes, such as MMP2. An increase was observed in secretion of pro-angiogenic factors including IGFBP2, IL1β, IL6, IL18, HGF and TNF-α by MSC-EVs and MSC-CM, and anti-fibrotic cytokines such as IFN-γ, IL10, IL27 by conditioned HUBFs.

Mesenchymal Stem/Stromal Cell (MSC) Culture and Characterization
Human mesenchymal stem/stromal cells (MSCs) were isolated from bone marrow aspirates, obtained from Lonza (Walkersville, MD, USA, WV IM-105), sourced from healthy (BMI < 25, non-diabetic) informed consent donors aged 30-40 years. Ten millimeters of unprocessed human bone marrow contained an average of 20 × 10 6 nucleated cells/mL. Human cells were seeded at a density of 0.16 × 10 6 cell/cm 2  Cultures were kept at 37 • C, 5% CO 2 , and 95% humidity. Twenty-four hours later, non-adherent cells were withdrawn, and refreshment with mesenchymal stem cell growth medium was carried out. Culture medium was changed 3 times per week, until 80% confluence was obtained. At this stage, the cells are said to be at passage 0. At each phase of expansion, MSCs were seeded at a density of 5000 cells per cm 2 . During this study, the cells were amplified up to passage 3 (P3), to limit any phenotypic change in the primary cells. At P3, their differentiation potential and phenotype were analyzed following International Society for Cell and Gene Therapy (ISCT) recommendations [13,14]. For phenotyping, the cells were labeled with the CD90-FITC, CD105-FITC, CD73-PE, CD34-PE and CD45-PE antibodies (BD Biosciences) and analyzed by flow cytometry. For differentiation potential, MSCs were cultured with lineage-specific media, StemPro Adipogenesis Differentiation Kit (Gibco, A10070-01), StemPro Osteogenesis Differentiation Kit (Gibco, A10072-01) StemPro Chondrogenesis Differentiation Kit (Gibco, A10071-01) for 21 days to induce cell differentiation. Differentiation into adipocytes, osteocytes and chondrocytes was detected by Oil Red O, alizarin red and alcian blue, respectively, and observed upon visible light microscopy.

Isolation of MSC-EVs et MSC-CM Collection
At passage 3 of MSCs, (80% of confluence) the cells were washed at least three times with sterile 1× PBS (Gibco) and re-cultured in serum-free growth medium for 72 h. Then, the medium was collected and centrifuged (3000× g, 10 min) to remove whole cells and debris. A part of the supernatant was kept at −80 • C (MSC-CM), and the other part was filtered by tangential flow filtration (TFF) using syringes with single-use hollow fiber filters (Repligen, mPES 500k) to isolate and concentrate MSC-EVs ( Figure 1). Quantification and sizing of particles present in MSC-CM and MSC-EVs samples was carried out using the NanoSight NS300 instrument (Malvern Panalytical). CD63 levels, widely used as EVs markers, were measured, in MSC-CM and MSC-EVs, using the ExoELISA-ULTRA CD63 kit (System Biosciences, Palo Alto, CA, USA) according to the manufacturer's protocol. EVs were visualized by cryo-electron microscopy (Cryo-EM). For the sample preparation, Quantifoil grids R1.2/1.3 from Agar Scientific were glow-discharged (20 s, 5 mA) using a Leica EM ACE600 system. Then, 2.5 µL of vesicles in PBS (1.4 × 10 9 EV/mL) was deposited on the carbon side of the EM grid, blotted for 1s with a blot force of 2 and plunge-frozen in liquid ethane using a Vitrobot Mark IV (ThermoFisher, Waltham, MA, USA). For the cryo-EM analysis, a Titan Krios 300KV cryo-electron microscope was used, equipped with a Falcon III direct electron detector (FEI, Hillsboro, OR, USA) and CMOS CETA detector (FEI, Hillsboro, OR, USA). The images were taken on the CETA detector at a magnification of 37,000× with a pixel size of 0.23 nm, a defocus of −2 µm using an aperture of 100 µm. The exposure time of the images was 1 s with an accumulated total dose of 40 e-/Å 2 per image. No filtering procedures were applied to images. filtered by tangential flow filtration (TFF) using syringes with single-use hollow fiber filters (Repligen, mPES 500k) to isolate and concentrate MSC-EVs ( Figure 1). Quantification and sizing of particles present in MSC-CM and MSC-EVs samples was carried out using the NanoSight NS300 instrument (Malvern Panalytical). CD63 levels, widely used as EVs markers, were measured, in MSC-CM and MSC-EVs, using the ExoELISA-ULTRA CD63 kit (System Biosciences, California, CA, USA) according to the manufacturer's protocol. EVs were visualized by cryo-electron microscopy (Cryo-EM). For the sample preparation, Quantifoil grids R1.2/1.3 from Agar Scientific were glow-discharged (20 s, 5 mA) using a Leica EM ACE600 system. Then, 2.5 μL of vesicles in PBS (1.4 × 10 9 EV/mL) was deposited on the carbon side of the EM grid, blotted for 1s with a blot force of 2 and plunge-frozen in liquid ethane using a Vitrobot Mark IV (ThermoFisher, Massachusetts, MA, USA). For the cryo-EM analysis, a Titan Krios 300KV cryo-electron microscope was used, equipped with a Falcon III direct electron detector (FEI, Hillsboro, OR, USA) and CMOS CETA detector (FEI, Hillsboro, OR, USA). The images were taken on the CETA detector at a magnification of 37,000× with a pixel size of 0.23 nm, a defocus of −2 μm using an aperture of 100 μm. The exposure time of the images was 1 s with an accumulated total dose of 40 e-/Å 2 per image. No filtering procedures were applied to images. Cell-free supernatants divided into two parts. One part corresponding to MSC-CM (4). The second part filtered by tangential filtration for isolated MSCEV (5) and analyzed (6). (7) Banking of MSC-EV.

Analysis of the Angiogenic Potential of MSC-EVs and MSC-CM
Protein contents of MSC-EVs and MCS-CM samples were measured using a BCA protein assay kit (Thermo Scientific Pierce, Rockford, IL, USA). The expression of angiogenic protein was analyzed using Proteome Profiler Human Angiogenesis Array (R&D Systems, catalogue number ARY007). Briefly, MCS-EVs and MCS-CM were mixed with the biotinylated detection antibody cocktail and incubated with nitrocellulose membranes containing spotted specific antibodies. Following adsorption of the biotinylated-antibody-antigen complexes, membrane was washed and further incubated with Streptavidin-HRP for quantification. Cytokine detection was performed using a ChemiDoc XRS+ System (BioRad, California, CA, USA). The signal density of each blot was quantified by dot blot analysis on NIH ImageJ analysis software 1.8.0. The level of 3 pro-angiogenic (1) expansion, (2-3) obtain cell-free supernatants, after cell culture in serum-free medium (72 h). Cell-free supernatants divided into two parts. One part corresponding to MSC-CM (4). The second part filtered by tangential filtration for isolated MSCEV (5) and analyzed (6). (7) Banking of MSC-EV.

Analysis of the Angiogenic Potential of MSC-EVs and MSC-CM
Protein contents of MSC-EVs and MCS-CM samples were measured using a BCA protein assay kit (Thermo Scientific Pierce, Rockford, IL, USA). The expression of angiogenic protein was analyzed using Proteome Profiler Human Angiogenesis Array (R&D Systems, catalogue number ARY007). Briefly, MCS-EVs and MCS-CM were mixed with the biotinylated detection antibody cocktail and incubated with nitrocellulose membranes containing spotted specific antibodies. Following adsorption of the biotinylated-antibodyantigen complexes, membrane was washed and further incubated with Streptavidin-HRP for quantification. Cytokine detection was performed using a ChemiDoc XRS+ System (BioRad, California, CA, USA). The signal density of each blot was quantified by dot blot analysis on NIH ImageJ analysis software 1.8.0. The level of 3 pro-angiogenic proteins was measured with a Quantikine ELISA kit (R&D systems): IL8 (D8000C), MCP-1 (DCP00) and CXCL16 (DCX160), following the manufacturer's instructions. Fluorescence images were captured at a 10× magnification using CKX53 brightfield and epifluorescence microscope (Olympus, Allentown, PA, USA). Number of nodes, number of junctions and segment length were counted from the collected images using the NIH ImageJ analysis software, to quantify the angiogenic network formation.

RNA Extraction and Reverse Transcription, Quantitative Real-Time Polymerase Chain Reaction to Analyze Fibrosis Genes
From D2 to D7, the culture supernatants and the cell pellets were harvested and stored at −80 °C. RNA was extracted from these pellets using NucleoSpin ® RNA Set for NucleoZOL (Macherey-Nagel, ref 740406, Hoerdt, France). Briefly, after lysing the sample in NucleoZOL and pelleting contaminating DNA and proteins, the supernatant was mixed with an optimized binding buffer for efficient binding of small and large RNA to the silica membrane. RNA was quantified using a nanodrop. Reverse transcription of 1 μg total RNA per reaction was carried out using iScript cDNA Synthesis Kit (BIO RAD

HUBFs Preconditioned with MSC-EVs or MSC-CM
Seventy-two hours before irradiation, the HUBFs were inoculated in 6-well plates at a density of 50,000 cells per well. They were incubated at 37 • C either in 2 mL complete medium for fibroblasts alone, or in 2 mL complete medium for fibroblasts contain-

RNA Extraction and Reverse Transcription, Quantitative Real-Time Polymerase Chain Reaction to Analyze Fibrosis Genes
From D2 to D7, the culture supernatants and the cell pellets were harvested and stored at −80 • C. RNA was extracted from these pellets using NucleoSpin ® RNA Set for NucleoZOL (Macherey-Nagel, ref 740406, Hoerdt, France). Briefly, after lysing the sample in NucleoZOL and pelleting contaminating DNA and proteins, the supernatant was mixed with an optimized binding buffer for efficient binding of small and large RNA to the silica membrane. RNA was quantified using a nanodrop. Reverse transcription of 1 µg total RNA per reaction was carried out using iScript cDNA Synthesis Kit (BIO RAD RNA) according to the manufacturer's specifications ( Figure 2).
TaqMan gene expression assays (Life Technologies; Applied Biosystems, Waltham, MA, USA) were used to quantify the following transcripts: α-SMA (Hs00426835_g1), CTGF (Hs00170014_m1), TIMP1 (Hs01092512_g1), Col3α1, (Hs00943809_m1), COL1α2 (Hs01028956_m1), MMP2 (Hs01548727_m1), TGF-β1 (Hs00998133_m1. Gene expression levels were normalized to GADPH (Hs02786624_g1), which was used as reference gene for HUBF cells. Corresponding to mRNA, cDNA was used at a concentration of 25 ng/mL. qRT-PCR was performed with cDNA from 50 ng total RNA and TaqMan Fast Universal Master Mix (Applied Biosystems, Waltham, MA, USA) on a StepOnePlus instrument (Applied Biosystems, Waltham, MA, USA) under the following standard conditions: 95 • C for 20 s, followed by 40 cycles of 95 • C for 1 s and 60 • C for 20 s. The relative gene expression was calculated via the comparative Ct method as previously described by K. Livak (Applied Biosystems User Bulletin #2, 2001, Waltham, MA, USA). Ct values were normalized to GADPH and used to calculate the relative gene expression using the 2 −∆∆Ct method. The fold change was calculated relative to the gene expression of non-irradiated (NIR) HUBFs.

Analysis of HUBF Secretome Cytokine Profile
The cytokine profile was analyzed using Proteome Profiler Human XL Cytokine Array (R&D Systems Catalog Number ARY022B). Briefly, the HUBF secretome was mixed with the biotinylated detection antibody cocktail and incubated with nitrocellulose membranes. The washed membranes were incubated with Streptavidin-HRP. Cytokine detection was performed using a ChemiDoc XRSn+ System (BioRad, Hercules, CA, USA). The signal density of each blot was quantified by dot blot analysis on the NIH ImageJ analysis software.

Statistical Analysis
To determine the effect of radiation exposure, MSC-EVs and MSC-CM on the phenotype and activation of HUBFs into myofibroblasts, variations in gene expression were compared. Statistical significance was calculated using the Mann-Whitney test. Significance for all analyses was set at p-value < 0.05 (*), p value < 0.01 (**) and p value < 0.001 (***). We used GraphPad Prism 7.0 software (https://graphpad-prism.software.informer.com/7.0/). All values were expressed as the Mean and SEM. Each condition consisted of 6-8 samples of HUBFs.
The mRNA level of GAPDH gene was considered as normalizing control gene. We interpreted the fold changes as follows: If there is a doubling (fold change = 2, Log2FC = 1) between IR and NIR, then IR is twice as large as NIR (or IR is 200% of NIR).
If there is a two-fold decrease (factor change = 0.5, Log2FC = −1) between IR and NIR, then IR is half as large as NIR or IR is 50% of NIR.
In this study, we consider a significant overexpression from 1.5 FC and underexpression from 0.5 FC of IR versus NIR.

EVs and MCS-CM Characterization
The size and concentration of extracellular vesicles in samples were measured by Nanoparticle Tracking Analysis (NTA). NTA demonstrated a comparable particle size distribution between MSC-CM and MSC-EV samples, of 114 and 121 nm, respectively (mode, n = 5). These results are confirmed by the observations of vesicle morphology made by means of cryo-electron microscopy. The mean concentration of particles measured by NTA was 2.63 × 10 8 /mL in MSC-CM samples and 1.45 × 10 9 in MSC-EV samples. This result shows that tangential filtration concentrates the vesicles because they are more numerous in MSC-EVs. Tangential filtration significantly increases by a factor of 5.5 (p = 0.0159). The level of CD63, a specific marker for EVs, proves that EVs are present in both MSC-CM and MSC-EVs preparations and that they are more concentrated in MSC-EVs ( Figure 4).

EVs and MCS-CM Characterization
The size and concentration of extracellular vesicles in samples were measured by Nanoparticle Tracking Analysis (NTA). NTA demonstrated a comparable particle size distribution between MSC-CM and MSC-EV samples, of 114 and 121 nm, respectively (mode, n = 5). These results are confirmed by the observations of vesicle morphology made by means of cryo-electron microscopy. The mean concentration of particles measured by NTA was 2.63 × 10 8 /mL in MSC-CM samples and 1.45 × 10 9 in MSC-EV samples. This result shows that tangential filtration concentrates the vesicles because they are more numerous in MSC-EVs. Tangential filtration significantly increases by a factor of 5.5 (p = 0.0159). The level of CD63, a specific marker for EVs, proves that EVs are present in both MSC-CM and MSC-EVs preparations and that they are more concentrated in MSC-EVs ( Figure 4).

In Vitro Human Radiation Cystitis Model
We analyzed this fibrogenic differentiation of HUBFs after irradiation by SARRP, and reported mRNA expression profiles of myofibroblast-associated genes in HUBFs irradiated at day (D) 5 post irradiation compared to non-irradiated HUBF ( Figure 5). A significant increase in the gene expression of the pro-fibrosating genes at D5 was observed. The level of myofibroblast marker was significantly increased in irradiated HUBFs. Expression of α-SMA, CTGF, TGF-β1, TIMP1, TIMP2, Col3α1, Col1α2 and matrix metallopeptidases (MMPs) were upregulated in irradiated HUBFs compared to non-irradiated HUBF. At D5 post irradiation, a significant increase was observed in α-SMA (+7. 13 Figure 5). These results support the idea that the irradiation protocol leads, at D5 post irradiation, to an activation of HUBFs into myofibroblasts, which are responsible for the development of fibrosis.

Secretome Analysis of Irradiated HUBF Cells
The protein expression of the secretome of irradiated and non-irradiated HUBFs was analyzed using the Proteome Profiler XL cytokine Array Kit (Figure 6). At D5 post irradiation, this semi-quantitative analysis shows a significant increase in pro-fibrotic proteins, IGFBP2, IL1β, IL6, IL18, and TNFα and HGF, within the secretome of irradiated HUBFs compared to non-irradiated HUFBs. At the same time, anti-fibrotic proteins IFNγ, IL10 and IL27 decrease significantly compared to non-irradiated fibroblasts. The HUBFs gene profile at D5 post irradiation with this molecular signature of fibrotic tissue indicates that the radiation-induced bladder myofibroblasts are in a pro-fibrotic environment. This in vitro model aims to represent radiation-induced myofibroblasts and their autocrine and paracrine environment at the stage of radiation cystitis, in order to examine cell-based responses, therapeutic responses of MSC-EVs and MCS-CM after radiation exposure.

Secretome Analysis of Irradiated HUBF Cells
The protein expression of the secretome of irradiated and non-irradiated HUBFs was analyzed using the Proteome Profiler XL cytokine Array Kit (Figure 6). At D5 post irradiation, this semi-quantitative analysis shows a significant increase in pro-fibrotic proteins, IGFBP2, IL1β, IL6, IL18, and TNFα and HGF, within the secretome of irradiated HUBFs compared to non-irradiated HUFBs. At the same time, anti-fibrotic proteins IFNγ, IL10 and IL27 decrease significantly compared to non-irradiated fibroblasts. The HUBFs gene profile at D5 post irradiation with this molecular signature of fibrotic tissue indicates that the radiation-induced bladder myofibroblasts are in a pro-fibrotic environment. This in vitro model aims to represent radiation-induced myofibroblasts and their autocrine and paracrine environment at the stage of radiation cystitis, in order to examine cell-based responses, therapeutic responses of MSC-EVs and MCS-CM after radiation exposure.

MSC-EVs and MCS-CM Dowregulate the Expression of Pro-Fibrotic Genes and Up-Regulate Anti-Fibrotic Genes in Irradiated HUBF Cells
MSC-EVs and MCS-CM changed the phenotype of irradiated HUBFs. Indeed, α-SMA gene expression in irradiated HUBFs preconditioned with MSC-EVs and MCS-CM was 3.85 FC ± 0.43 and 5.86 FC ± 0.58, respectively, compared to non-irradiated HUBFs. These expression levels are largely reduced compared to irradiated fibroblasts (+7.13 FC ± 0.96). In particular, when the fibroblasts are preconditioned with MSC-EVs, a significant decrease by a factor of 2 (p = 0.0041) is observed compared to irradiated HUBFs (Figure 7). Likewise, CTGF gene expression of irradiated fibroblasts preconditioned with MSC-EVs and MCS-CM was 2.77 FC ± 0.16 and 0.75 FC ± 0.10, respectively, compared to non-Irradiated HUBFs. These expression levels are notably reduced for the two preconditionings compared to irradiated HUBFs (10.96 FC ± 0.19). The expression level of irradiated HUBFs preconditioned with MSC-EVs is significantly reduced compared to irradiated HUBFs by a factor of 4 (p < 0.001). Additionally, the expression level of irradiated HUBFs preconditioned with MCS-CM is significantly reduced by a factor of 15 (p < 0.001) compared to irradiated HUBFs. Preconditioning with MCS-CM results in a significant decrease in CTGF gene expression by a factor of 4 (p < 0.001) compared to irradiated HUBFs preconditioned MSC-EVs ( Figure 7).
Next, the effect of MSC-EVs and MCS-CM on the gene expression of extracellular matrix components such as type I (Col1α2) and type III (Col3α1) collagen was analyzed (Figure 7).
Col1α2 gene expression of irradiated HUBFs preconditioned with MSC-EVs and MSC-CM was 15.39 ± 4.54 and 8.01 ± 1.36, respectively, compared to non-irradiated HUBFs. This expression level is significantly reduced with MC-MSC preconditioning by a factor of 2.7 (p < 0.001) compared to irradiated HUBFs (21.62 FC ± 3.26). No significant difference was found between the two preconditionings ( Figure 7).
MMP2 gene expression of irradiated HUBFs preconditioned with MSC-EVs and MSC-CM was 6.17 ± 0.47 and 7.86 ± 1.41, respectively, compared to non-irradiated HUBFs. The increase in these expression levels is not significant compared to irradiated fibroblasts (5.02 FC ± 0.74) due to a large degree of variability (Figure 7).
These results seem to suggest an almost equivalent anti-fibrotic potential of MSC-EVs and MSC-CM. MSC-EVs and MSC-CM also take part in the wound-healing process after irradiation while inhibiting fibrosis by maintaining production of Col3α1, TIMP1, and TGF-β1. Pre-conditioning before irradiation did not lead to variation in Col3α1 gene expression by irradiated fibroblasts. Col3α1 gene expression of the HUBFs preconditioned with MSC-EVs and MSC-CM was 75.35 ± 12.43 and 77.60 ± 9.51, respectively, compared to non-irradiated HUBFs (+57.23 FC ± 1.85). This increase in the expression level is not significant compared to irradiated HUBFs (Figure 7). Overexpression of Col3α1 by non-irradiated HUBFs, in order to maintain homeostasis was notable. Tissue inhibitors of metalloproteinases (TIMPs) are tissue specific, endogenous inhibitors of metalloproteinases, including matrix metalloproteinases (MMPs). The TIMP1 gene expression of irradiated HUBFs preconditioned with MSC-EVs and MSC-CM was 3.22 ± 0.18 and 6.86 ± 1.20, respectively, compared to non-irradiated HUBFs. The expression level of TIMP1 is significantly increased by the two preconditionings compared to irradiated HUBFs (+2.09 FC ± 0.14). Preconditioning with MSC-CM appears to be significantly greater by a factor of 2 (p = 0.0226) compared to those preconditioned with MSC-EVs (Figure 7). TGF-β1 gene expression of irradiated HUBFs preconditioned with MSC-EVs and MSC-CM was 4.23 ± 0.59 and 7.51 ± 0.94, respectively, compared to non-irradiated HUBFs. The expression level of TGF-β1 was significantly increased for the two preconditionings compared to irradiated HUBFs (2.41 FC ± 0.25). This elevation of TGFβ1 gene expression of HUBFs preconditioned with MSC-CM appears to be significantly greater by a factor of 2 (p = 0.0218) compared to HUBFs preconditioned by MSC-EVs before irradiation (Figure 7). These results underline that MSC-EVs and MSC-CM also contribute to the wound-healing process following irradiation while inhibiting fibrosis by maintaining Col3α1, TIMP1 and TGF-β1 production.

MSC-EVs and MSC-CM Modulate the Secretome of Irradiated HUBFs
The protein expression of the secretome in irradiated HUBFs pre-conditioned with MSC-EVs and MSC-CM was analyzed using the Proteome Profiler Human XL cytokine Array Kit and compared to the protein expression of the secretome in irradiated HUBFs (Figure 8). At D5 post irradiation, this semi-quantitative analysis shows a significant decrease in pro-fibrotic proteins within the secretome of irradiated HuFBs pre-conditioned with MSC-CM and MSC-EVs compared to irradiated HUBFs: IGFBP2, IL1-β, IL6, IL11, IL18, PDGF, TNFα, HGF in MSC-CM, MSC-EVs and irradiated HUBF groups, respectively. Furthermore, a significant increase was observed in anti-fibrotic proteins within the secretome of irradiated HUBFs preconditioned with MSC-CM and MSC-EVs compared to irradiated HUBFs: with IFNγ, IL10 and IL27 in MSC-CM, MSC-EVs and irradiated HuFB groups, respectively. HUBFs: IGFBP2, IL1-β, IL6, IL11,  IL18, PDGF, TNFα, HGF in MSC-CM, MSC-EVs and irradiated HUBF groups, respectively. Furthermore, a significant increase was observed in anti-fibrotic proteins within the secretome of irradiated HUBFs preconditioned with MSC-CM and MSC-EVs compared to irradiated HUBFs: with IFNγ, IL10 and IL27 in MSC-CM, MSC-EVs and irradiated HuFB groups, respectively. This points to an anti-fibrotic potential for the two preconditionings, MSC-EVs and MSC-CM, without evidence of a stronger potential in one or the other.

Potential Pro-Angiogenic of MSC-EVs and MSC-CM
The expression of proteins involved in angiogenesis was analyzed for MSC-EVs and MSC-CM using the Proteome Profiler Human Angiogenesis Array Kit ( Figure 9A). This semi-quantitative analysis shows an overexpression of pro-angiogenic proteins by MSC-EV and MSC-CM, such as VEGF, IGFβ2 and IGFβ3 ( Figure 9B). Quantification by ELISA also shows overexpression of three proteins essential to angiogenesis, IL-8, MCP1, and CXCL16 ( Figure 9C). These data confirm the proangiogenic potential of MSC-EVs and MSC-CM. MSC-EVs seem to express these proteins in greater quantity than MSC-CM. This points to an anti-fibrotic potential for the two preconditionings, MSC-EVs and MSC-CM, without evidence of a stronger potential in one or the other.

Potential Pro-Angiogenic of MSC-EVs and MSC-CM
The expression of proteins involved in angiogenesis was analyzed for MSC-EVs and MSC-CM using the Proteome Profiler Human Angiogenesis Array Kit ( Figure 9A). This semi-quantitative analysis shows an overexpression of pro-angiogenic proteins by MSC-EV and MSC-CM, such as VEGF, IGFβ2 and IGFβ3 ( Figure 9B). Quantification by ELISA also shows overexpression of three proteins essential to angiogenesis, IL-8, MCP1, and CXCL16 ( Figure 9C). These data confirm the proangiogenic potential of MSC-EVs and MSC-CM. MSC-EVs seem to express these proteins in greater quantity than MSC-CM.
In order to evaluate the angiogenic potential of MSC-CM and MSC-EVs, endothelial tube formation assays were performed. For the quantification using the NIH ImageJ analysis software, we used three indicators to determine the angiogenic effects of MSC-CM and MSC-EVs: the number of nodes, the number of junctions, and the segments length. As illustrated in Figure 10, HUVECs from incubation with MSC-CM and with MSC-EVs formed more nodes, junctions, and longer segments than the cells incubated with medium only. In order to evaluate the angiogenic potential of MSC-CM and MSC-EVs, endothelial tube formation assays were performed. For the quantification using the NIH ImageJ analysis software, we used three indicators to determine the angiogenic effects of MSC-CM and MSC-EVs: the number of nodes, the number of junctions, and the segments length. As illustrated in Figure 10, HUVECs from incubation with MSC-CM and with MSC-EVs formed more nodes, junctions, and longer segments than the cells incubated with medium only.

Discussion
To the best of our knowledge, our study is the first to develop a model of radio-induced bladder myofibroblasts in a pro-fibrotic environment in vitro. We have also demon-

Discussion
To the best of our knowledge, our study is the first to develop a model of radio-induced bladder myofibroblasts in a pro-fibrotic environment in vitro. We have also demonstrated that MSC-EVs and MSC-CM are two potential candidates in the prevention of radiation cystitis, due to their capacity to inhibit the bladder myofibroblast phenotype, their capacity to decrease the secretion of pro-inflammatory cytokines of HUBFs and, finally, their proangiogenic capacities to help maintain vascular integrity and limit tissue ischemia.
Acute radiation tissue injury to the bladder is characterized by edema, hyperemia and inflammation of the mucous membrane. Urothelium is fragile and vulnerable to trauma and infections [16]. In late radiation tissue injury, the submucosal vascularity is damaged by fibrosis of the vascular intima resulting in vessel obliteration and submucosal/muscular fibrosis. This is followed by urothelial atrophy, hypoxia with hypovascularization and ischemia of the bladder leading to the development of fibrosis and atrophy of the bladder tissue [1,17]. Clinical symptoms may include increased urinary urgency and frequency (pollakiuria), both during the day and at night, dysuria, but also cystalgia with bladder spasms, and hematuria. These adverse events have a serious impact on patients' quality of life and can be life-threatening in some cases. Nowadays, their management is limited to symptomatic treatment and remains a therapeutic challenge.
To date, the pathophysiology of RC remains unclear. However, it has been established that the bladder fibroblast plays a key role in the process of fibrosis, by its role in the overproduction of the extracellular matrix but also by its interaction with the immune system and endothelial cells [18].
Our work initially involved establishing a model of RC in vitro (bladder myofibroblasts activated in a radio-induced pro-fibrotic environment) so as to assess the effect of irradiation on the fibroblast and its implication in these three key stages of post-radiation bladder fibrosis.
In our study, a 3.5 Gy dose of radiation per day for 3 consecutive days using SARRP was applied to create an in vitro model of radiation cystitis. Our data demonstrated significantly increased mRNA expression of α-SMA, CTGF, Col1α2, Col3α1, TGF-β at D5. Indeed, CTGF had previously been reported to modulate many signaling pathways responsible for tissue remodeling and fibrosis development [19]. CTGF protein induces gene expression and levels of proteins involved in the synthesis of the extracellular matrix (ECM) [20].
TGF-β is the primary factor that drives fibrosis [21]. Activation of TGF-β signaling, for example by fibroblast-specific overexpression of constitutively active TGF-β receptor type 1 (TGF-β-RI), is sufficient to induce a systemic fibrotic disease with progressive fibrosis in multiple tissues [22]. TGF-β has a close association with Matrix MetalloProteinases (MMPs), a family of proteolytic enzymes involved in the degradation and remodeling of extracellular matrix proteins.
Tissue inhibitors of metalloproteinases 1 (TIMP1) expression is increased within these profibrotic environments, which suggests a role for TIMP1 in restricting ECM proteolysis [19,23]. McLennan et al. reported a close interaction between CTGF and TGF-β and TIMP1 in the development of fibrosis in the context of hyperglycemia. However, above all, maintaining a high level of CTGF is necessary to sustain the process of fibrosis [20]. CTGF is therefore one of the essential therapeutic targets in the prevention of fibrosis [24]. Additionally, maintaining the correct level of TGF-β ensures healing and homeostasis while avoiding autoimmune pathologies [18,25,26].
An excess in collagen production is necessary for the development of pulmonary fibrosis. In the pulmonary fibrosis model, less-elastic type-I collagen was found to predominate, perhaps even exclusively [27,28].
These pathological features were consistent and provided a basis for the successful establishment of an in vitro radiation cystitis model. In addition, analysis of irradiated fibroblasts secretome at D5 revealed a molecular signature of fibrotic tissue, with increased secretion of pro-fibrotic cytokines, such as IGFBP2, IGFBP3, IL1b, IL4, IL6, IL 11, IL18, IL33,  PDGF and TNFα, and a decrease in anti-fibrotic molecules, such as IL10. IL-1β and TNFα are pro-inflammatory cytokines that are expressed in common chronic pathologies such as rheumatoid arthritis, diabetes, myocardial infarction, and inflammatory lung diseases [29]. IL-10 is a potent anti-fibrotic and immunomodulatory cytokine secreted in response to elevated levels of inflammatory cytokines such as IL-β, TNFα, and interferon (IFN) [30,31].
This modification of the secretome of irradiated HUBF on line the bystander effects of ionizing radiation (the non-targeting effects irradiation), and thus promotes the fibrosis process. This bystander effect has been reported in other in vitro models, notably in the process of angiogenesis [32].
Other molecules are also involved in the fibrosis process such as MMP9 and TIMP2. Indeed, Mohammed and Said reported that MMP-9 is constitutively expressed in the intestinal mucosa, and is highly expressed in rat intestines after Υ-radiation exposure, and its inhibition leads to a tendency to inhibit fibrosis [33]. Wang et al. confirm the role of MMP9 in the process of liver fibrosis, but its role is dynamic over time [34]. Additionally, the different properties of MMP9 must continue to be explored.
In our model, we did not find any significant evolution of MMP9 and TIMP2 gene expression between irradiated and non-irradiated HUBF. Zwaans et al. report no difference in PAI, TIMP1 and TIMP2 expression in urine between prostate cancer patients who underwent pelvic irradiation. Similarly, there was no significant difference in the expression of PAI, TIMP1 and TIMP2 in long-surviving patients with CR symptoms compared to patients who also received pelvic radiotherapy but did not show CR symptoms [35]. Thus, we need further investigation to determine the place of these molecules in the radiation cystitis model. This in vitro model allows for an initial assessment of new anti-fibrotic therapies before studying their effectiveness in vivo, in accordance with the "3R" principles of bioethics: Reduce, Refine and Replace.
The use of MSCs in tissue remodeling and repair has grown notably in recent years. Few studies have looked into the place of MSCs in the management of radiation cystitis. Wiafe et al. reported that co-culture of MSCs with bladder smooth muscle cells (bSMCs) potently mitigates hypoxia-induced inflammatory and profibrotic pathways, with >50% inhibition of hypoxia-induced TGFβ1 and IL-6 expression (p < 0.005) and increased IL-10 protein (p < 0.005) [29]. Moreover, Qiu et al. have demonstrated the protective effect of adipose-derived mesenchymal stem cells (AdMSCs) in the management of radiationinduced bladder dysfunction and histological changes in an in vivo model. These results suggest a potential for MSCs in the management of radiation cystitis [36]. However, their clinical application remains limited, partly due to the difficulty of isolating them and obtaining quantities sufficient for the management of these pathologies [1,8,37].
MSC-EVs and MSC-CM appear to be therapeutic candidates in tissue remodeling and repair.
EVs, including exosomes and microvesicles, are a heterogeneous population of lipid membrane-delimited nanoparticles encapsulating a plethora of bioactive factors, including proteins (cytokines, membrane receptors, growth factors and enzymes) and genetic materials (mRNAs and microRNAs) [38,39]. They function as key intercellular signaling mediators to elicit biological responses via horizontal transfer of their bioactive cargo into recipient cells [38][39][40]. A further advantage, compared with the original MSCs, is that MSC-EVs cannot self-replicate, thus allaying safety concerns associated with cell therapy, such as uncontrolled cell division and cellular contamination with tumorigenic cells [10,41]. Likewise, conditioned medium from human mesenchymal stromal cells (MSC-CM) has demonstrated therapeutic potential in various fields such as myocardial infarction, stroke and acute and chronic hindlimb ischemia, neurodegenerative diseases, spinal cord injury, alopecia, acute and chronic wounds, acute liver injury/failure, lung injury, periodontal tissue injury, male infertility, soft tissue and bone defects [21][22][23][24][25][26]. Different studies have been conducted involving the use of MSC-CM for hair follicle regeneration, fractionated carbon dioxide wound healing, and treatment of inflammatory arthritis and multiple sclerosis [42][43][44][45][46]. Thus, the MSC secretome has been suggested as a novel cell-free medicinal product that can recapitulate the beneficial effects of MSCs and has various advantages in overcoming the limitations and risks associated with cell-based therapy [40,41].
We have demonstrated that MSC-EVs and MSC-CM are candidates in the prevention of radiation cystitis with the potential to inhibit the fibrotic process, inhibit the myofibroblast phenotype, decrease the secretion of pro-fibrotic cytokines and increase the secretion of antifibrotics cytokines by HUBF cells, and finally through their own pro-angiogenic properties.
First of all, MSC-EVs and MSC-CM inhibit the myofibroblastic phenotype and, therefore, decrease the production of the extracellular matrix, the first key step in radiation fibrosis. Indeed, the gene expression of αSMA, CTGF and Col1α2, which play an essential role bladder fibrosis, was found to decrease.
At the same time, an increase was observed in the gene expression of MMP2. The increase in MMP2 and the decrease in Col1α2 are responsible for the degradation of fibrillar collagen [47].
MSC-EVs and MSC-CM do not influence gene expression of Col3α1, TIMP1 and TGFβ. Col3α1 and TIMP1 are involved in the wound remodeling phase and ensure homeostasis. TIMP1 is also involved in restricting both inflammation and ECM accumulation/fibrosis following injury [23]. Timp1 −/− mice had significantly increased injury, inflammation, and fibrosis following carbon tetrachloride-induced liver injury compared to wild type mice. These studies provide evidence of a role for TIMP1 in restricting both inflammation and ECM accumulation/fibrosis following injury [48].
Based on these results, it appears that the effect of MSC-EVs and MSC-CM on HUBFs does not shift the balance towards a pro-fibrotic phenotype but rather to a pro-healing phenotype. Indeed, the persistence of TGF-β1, TIMP1 and Col3α1 contributes to the wound-healing process following irradiation. Tutuianu et al. confirm these results in an in vitro wound-healing model. They reported that the regenerative properties of MSCderived exosomes were validated using a wound-healing skin organotypic model, which exhibited full re-epithelialization upon exosomes exposure [47].
Basalova et al. demonstrated that MSC-EVs down-regulated secretion of extracellular matrix proteins by fibroblasts as well as suppressed their contractility, resulting in prevention as well as reversion of fibroblasts differentiation to myofibroblasts [49].
Moreover, MSC-EVs and MSC-CM induce a modulation of the secretome of irradiated HUBFs. Preconditioned HUBFs exhibit an anti-fibrotic secretome with inhibition of secretion of pro-fibrotic cytokines and increased secretion of anti-fibrotic proteins.
The proteins analyzed within the secretome have previously been identified as key proteins of fibrosis in clinical and preclinical studies [18,22,[50][51][52]. Zwaans et al. reported a high level of HGF in patients who received a diagnosis of radiation cystitis [36]. HGF could be one key element in the prevention of RC. We demonstrated a decrease in HGF secretion from preconditioned HUBFs.
Their action is possibly linked to their high concentration of miRNAs, such as miR-21 and miR-29, which mediate antifibrotic effects [49].
Finally, vascular lesion is the second essential step in the development of radiation cystitis, starting from the acute phase. Analysis of their composition reveals a pro-angiogenic and anti-inflammatory signature. We observed an increase in the level of IL-8 protein, which stimulates endothelial cell proliferation and capillary tube organization and promotes angiogenesis by directly interacting with endothelial cells [53]. MCP-1 can also act directly on endothelial cells to induce angiogenesis [54]. Human umbilical vein endothelial cells (HUVECs) treated with MCP-1 showed significantly increased numbers of capillarylike tube formation. CXCL16 is a robust agonist of both angiogenesis (HUVEC migration and incorporation) and vasculogenesis which promotes HUVEC proliferation, chemotaxis, and tube formation by activating ERK pathway in vitro [55]. Moreover, we demonstrated that HUVECs from incubation with MSC-CM and with MSC-EVs formed more nodes, junctions, and longer segments than the cells incubated with medium only.
These data confirm the therapeutic potential of MSC-EVs and MSC-CM in the management of tissue repair, and in our case the prevention of radiation cystitis.
As of today, MSC-EVs and MSC-CM are being evaluated in phase I/II clinical trials for the prevention or management of severe forms of COVID-19, and also inflammatory digestive pathologies, for their anti-inflammatory properties [55][56][57][58][59][60][61]. This confirms the feasibility of these new therapeutic possibilities in clinical practice, with intravenous but also intranasal delivery.
However, with equivalent therapeutic potential, MSC-CM seems easier to use. Indeed, the techniques necessary to isolate and characterize MSC-EVs are long and can be expensive [10,62,63]. This supports MSC-CM as a potential candidate for simpler and less expensive clinical application.

Conclusions
MSC-EVs and MSC-CM appear to offer a promising therapeutic approach for management of RC and thereby an opportunity to improve patients' the quality of life. In vitro, MSC-EV and MSC-CM appear to have promising therapeutic potential in the prevention of RC by targeting the three main stages of RC, fibrosis, inflammation and vascular damage.
If they have the same efficiency, then three important elements are to be emphasized on the conditioned medium of MSCs. MSC-CM is easily obtained in large quantities and can therefore be used locally by bladder instillation to locally reduce the development of radiation-induced fibrosis of the bladder. MSC-CM is richer in composition of proteins and other molecules, giving it a broader spectrum of action, or longer efficacy over time. Obtaining MSC-CM seems less expensive to obtain than MSC-EVs.
The efficacy and safety of these two approaches should be studied in a mouse model of radiation cystitis.

Data Availability Statement:
The data that support the findings of this study are available from the corresponding author upon request.